Capillary Electrophoresis (CE) has emerged as an important tool for analyzing biomolecules. The high efficiency, high resolution, and automation capabilities of CE make it highly suitable in the routine analysis of proteins, peptides, and even small ions. A major problem encountered in the above separations is the interaction of basic analytes, such as basic proteins, with exposed surface silanol groups on the capillary wall. This interaction results in a loss of efficiency and irreproducible separations. Typical approaches in addressing the above problem include working at conditions where the silanol groups are either un-ionized.sup.1 or fully ionized.sup.2. These conditions, however, entail working at extremes of pH and may be unsuitable for many analytes. Additionally, silica dissolves at extreme pH's, which is another limitation of this approach..sup.3 Other approaches in addressing the above problem involve adding compounds.sup.4-6 that compete with the analytes for interaction sites on the capillary wall. These additives, however, may adversely affect the separation of analytes.
Another popular approach includes working with coatings that are either physically adsorbed or chemically attached to the capillary surface..sup.7-21 These coatings mask the presence of surface silanols and enhance separation efficiency. The adsorbed coatings suffer from limited stability and require repeated replenishment for effective operation.sup.7. Recently, Gilges et al..sup.7 showed excellent separation of basic proteins using a polyvinylalcohol (PVA) coated capillary. The polymer coating was achieved by a thermal treatment that immobilized PVA on the capillary wall. This coated capillary gave a low electroosmotic (EO) flow up to pH 9. However, only 40 runs were possible at pH 8.5 without loss of efficiency. Buffers such as borate, Tris HCl, and Tri-phosphate did not provide good separation of proteins using this coated capillary, thus limiting its utility.
A review of coatings for CE reveals several examples of chemically modified capillaries that were designed to minimize the presence of surface silanols and reduce analyte interactions. These modifications involve attaching or creating one or more polymeric layers on the surface of the capillary through various coupling chemistries. In 1985, Hjerten.sup.8 showed a two-step coating process by attaching a bifunctional silane on the surface of the capillary followed by in situ polymerization of a vinyl group containing monomer. The presence of a polymerizable C.dbd.C group was essential in both the monomer and silane for coupling. Strege and Lagu.sup.9 showed that the above coating gives a very low EO flow, but achieved poor separations of a mixture of proteins. The poor peak shapes obtained with this capillary were attributed to electrostatic and/or hydrogen bonding interactions of the proteins with the capillary wall or coating. It was necessary to incorporate a surfactant in the CE run buffer to achieve good separations of proteins. Similarly, a cross-linked in situ polymerized polyacrylamide capillary gave poor separation efficiencies for basic proteins when tested with no added cationic additives in the buffer.sup.9.
As an alternative approach to in situ polymerization, coatings are formed by reacting silanes that have appropriate reactive end groups with reactive end groups on prederivatized polymers. These coatings were disclosed by Herren et al..sup.11 to minimize or reduce EO flow. They discussed several synthetic procedures for creating various derivatives of dextran.sup.12 and PEG.sup.13 and their utility in several applications i0ncluding modifying control pore glass beads. However, data on the pH stability of this coating and its performance with proteins as test analytes were not shown. Following a similar approach, Hjerten and Kubo.sup.14 showed the attachment of several polymers (e.g., methylcellulose and dextran) after a prederivatization step. The prederivatization step was required prior to attaching the polymers to the methacryl silane treated capillary. Additionally, the polymer coupling process was dependent upon a high yield of the prederivatization reaction.
Recently, Malik et al..sup.15,16 adapted a GC-type static coating procedure, in which the coating was achieved by depositing a mixture of polymer, initiators, and silane reagent on the surface of a capillary by using a low boiling point solvent The capillary was then heat treated to cross-link the surface film. The coating thickness influenced the EO flow and performance and required optimization. In comparing data from Malik et al.,.sup.15,16 variabilities in efficiencies were observed between analytes in a Superox-4 coated capillary and between two Superox-4 coated capillaries. Similarly, two Ucon 75-H-90000 polymer coated capillaries tested under identical conditions gave different migration times and mobilities, indicating problems with the reproducibility of the coating process.
The above coatings were attached through Si--O--Si--C linkage. To overcome the limited pH stability of the Si--O--Si bond, several researchers used approaches such as attaching polyacrylamide by in situ polymerization through a Si--C linkage.sup.17 and attaching a hydrolytically stable derivative of acrylamide by in situ polymerization..sup.18 These approaches enhanced the coating stability relative to Hjerten's original approach and provided better efficiencies for basic proteins. However, multiple reaction steps with stringent conditions were required during the coating process. For example, the approach by Cobb et al..sup.17 required anhydrous solvents and conditions during the Grignard reaction step. Similarly, the work by Chiari et al..sup.18 required synthesis of a special monomer to achieve a stable and efficient coating. Other approaches involved cross-linking or attaching several polymeric layers on the capillary surface. Increased coverage on the capillary surface by the various polymeric layers was expected to diminish any interaction of the analytes with the exposed surface silanols. Smith et al..sup.19 showed separations of proteins in coated capillaries that had a primary silane layer anchored to several polymeric layers. Some layers were adsorbed on top of the primary layer. Huang et al..sup.20 showed separation of proteins using a cross-linked, immobilized, hydrophilic polymer layer atop a hydrophobic, self assembled, alkyl silane layer. Schmalzing et al..sup.21 showed excellent separations of basic proteins in a multilayered cross-linked coated capillary. In situ polymerization of a monomer on top of a cross-linked primary silane layer resulted in a hydrophilic polymeric layer that was subsequently cross-linked. The above approaches were all multistep processes and, in some cases, required additional cross-linking steps..sup.21
There is a need for a simple method for coupling preformed underivatized polymers covalently to the surface of a conduit such as a fused silica capillary used for capillary electrophoresis.
In addition, polymer based support surfaces have been used for separating the components in a fluid stream such as for capillary electrophoresis or liquid chromatography. Such polymeric support surfaces can be on the inner walls of the conduit (e.g. capillary), or can form a packing of polymeric particles for liquid chromatography. In some instances, such polymeric support surfaces do not have the desired properties for separating components. For this purpose, such surfaces have been modified by coating with suitable hydrophilic polymers as disclosed in Afeyan et al. (U.S. Pat. No. 5,503,933).